Glycosylated analogs of formaecin I and drosocin exhibit differential pattern of antibacterial activity

Glycoconj J (2011) 28:537–555

DOI 10.1007/s10719-011-9353-2

Glycosylated analogs of formaecin I and drosocin

exhibit differential pattern of antibacterial activity

Sariya Talat & Menithalakshmi Thiruvikraman &

Saroj Kumari & Kanwal J. Kaur

Received: 12 August 2011 /Revised: 13 September 2011 /Accepted: 19 September 2011 /Published online: 4 October 2011

#

Springer Science+Business Media, LLC 2011

Abstract The synthetic glycopeptides are interesting model

systems to study the effect of O-glycosylation in modulating

their function and structure. A series of glycosylated analogs

of two antibacterial peptides, formaecin I and drosocin, were

synthesized by varying the nature of sugar and its linkage

with bioactive peptides to understand the influence of

structure variation of glycosylation on their antibacterial

activities. Higher antibacterial activities of all glycopeptides

compared to their respective non-glycosylated counterparts

emphasize in part the importance of sugar moieties in

functional implications of these peptides. The consequences

of the unique differences among the analogs were apparent

on their antibacterial activities but not evident structurally by

circular dichroism studies. We have shown that differently

glycosylated peptides exhibit differential effect among each

other when tested against several Gram-negative bacterial

strains. The change of monosaccharide moiety and/or its

anomeric configuration in formaecin I and drosocin resulted

into decrease in the antibacterial activity in comparison to

that of the native glycopeptide, but the extent of decrease in

antibacterial activity of glycosylated drosocin analogs was

less. Probably, the variation in peptide conformation arising

due to topological dissimilarities among different sugars in

the same peptide resulting in possible modulation in binding

properties appears to be responsible for differences in their

antibacterial activities. Indeed, these effects of glycosylation

are found to be sequence-specific and depend in the milieu

of amino acid residues. Interestingly, none of the carbohy-

drate variants affected the basic property of these peptides,

which is non-hemolytic and non-toxicity to eukaryotic cells.

.

Keywords Synthesis Glycosylated peptides Formaecin

.

I Drosocin Antibacterial activity

Abbreviations

CAMPs

Cationic antimicrobial peptides

TMSOTf Trimethylsilyl trifluoromethanesulfonate

DBU

HBTU

1,8-diazabicyclo[5.4.0]undec-7-ene

O-Benzotriazole-N,N,N’,N’-tetramethyl-uronium-

hexafluoro-phosphate

SDS

TFE

Sodium-dodecyl-sulfate

2,2,2-trifluoroethanol

Introduction

Carbohydrates are frequently a required component of

antibiotic agents that bind to either protein or RNA

target structures, or provide the requisite conformational

stability to them and such glycoconjugates therefore

have medicinal potential [1]. In general, protein glycosylation

is a ubiquitous post-translational modification capable of

transforming protein’s properties in different ways. It is

known that glycosylation can profoundly impact a protein’s

folding, stability, and intracellular trafficking [2–6]. The

structures and dynamics of several glycosylated peptides

have been elucidated and it has been shown that peptide

conformation is stabilized and its fluctuations restricted by

the glycosylation [4, 7–13]. However, a detailed understand-

ing of the functions of glycoproteins is often hindered by the

structural microheterogeneity caused by the diverse patterns

of glycosylation [3].

:

S. Talat M. Thiruvikraman S. Kumari K. J. Kaur (*)

Structural Biology Unit, National Institute of Immunology,

Aruna Asaf Ali Marg,

New Delhi 110 067, India

e-mail: kanwal@nii.res.in

538

Glycoconj J (2011) 28:537–555

In insects, glycosylation is one of the important post-

Results

translational modifications of short proline rich cationic

antimicrobial peptides (CAMPs) and glycosylated anti-

microbial peptides are found to be O-glycosylated.

Glycosylation seems to have important role in antimicro-

bial activity as removal of sugar moiety causes significant

reduction in activity. Drosocin [14], formaecins [15],

pyrrhocoricin [16] and lebocins [17] are examples of such

proline rich short CAMPs which are glycosylated at

threonine residue with an O-linked monosaccharide (α-

GalNAc) or a disaccharide (β-galactopyranosyl-α-

GalNAc) sugar moiety. Due to widespread resistance to

almost all conventional antibiotics, there is an urgent need

to discover or design novel antibiotics [18]. Naturally

occurring antimicrobial [19] and host defense peptides are

being developed as new anti-infective therapeutic agents

[20]. Peptide antibiotics offer several advantages such as

lower tendency to induce resistance in bacteria, specific

action against bacterial membranes, and the ability to

target both the surface and the intracellular milieu of

bacteria [20]. However, the mode of action of the proline

rich cationic antimicrobial peptides from the insects is not

known completely and they are thought to kill bacteria by

entering cells and binding target/targets to bring their

antibacterial activity [21–23]. With enormous potential

coded within in terms of configuration and nature,

carbohydrate molecules are quite an interesting tool for

development and designing of novel glycopeptide anti-

biotics. Indeed lesser understanding of sugar moiety in

relation to their coupling partners is a serious limitation in

their wide use as important conjugate in development of

sugar linked antibiotic agents.

Earlier it has been reported that O-glycosylation has

effect on the structure and biological activity of antibac-

terial peptides as compared to their non-glycosylated

counterparts [15, 17, 24–27]. We have shown that

conformation provided by carbohydrate can be mimicked

by strategically designed amino acids substitutions in the

native glycosylated antibacterial peptide and resulted into

a functionally equivalent non-glycosylated analog [25].

Here we examined the effect of different O-linked sugars

in two proline-rich glycosylated antibacterial peptides.

The possible consequences of variation in sugar residue

both in terms of its nature as well as configuration of

linkage to amino acid residue are determined in the

context of antibacterial activities. These variations are

studied in two different antibacterial glycopeptides, formaecin

I [GRPNPVNNKPT(αGalNAc)PHPRL] and M-drosocin

[GKPRPYSPRPT (αGalNAc)SHPRPIRV], which are having

differences in glycosylated threonine surrounding amino

acids. Secondary structure determination using circular

dichroism spectroscopy and cytotoxic activities of all the

peptides were also studied.

Synthesis of antibacterial glycopeptides with sugar variants

The proline rich class of antibacterial glycopeptides from

insects contain conserved threonine residue, which is

glycosylated by either a monosaccharide (2-acetamido-2-

deoxy-D-galactopyranosyl; GalNAc) or a disaccharide

(β-D-galactopyranosyl-GalNAc). These sugars are α-

linked to threonine. To explore the effect of variation

in the sugar moiety of antibacterial peptides, formaecin

I [GRPNPVNNKPT(αGalNAc)PHPRL] and M-drosocin

[GKPRPYSPRPT(αGalNAc)SHPRPIRV], on their structures

and antibacterial activities, various analogs having differences

in the sugar region and its linkage to peptide were synthesized.

The key challenge was the synthesis of differently

glycosylated threonine residues, which are the critical

building blocks for synthesizing various glycosylated analogs

of antibacterial peptides, formaecin I and M-drosocin. We

have also synthesized non-glycosylated analogs of native

formaecin I and M-drosocin to further analyze the influence of

sugar moiety on their functional acivities.

Various glycosylated analogs of threonine (Fig. 1) like

N^α-Fmoc-Thr(Ac₃-α-D-GalNAc)-OH (Ia), N^α-Fmoc-Thr

(Ac₃-α-D-GlcNAc)-OH (Ib), N^α-Fmoc-Thr(Ac₃-β-D-Gal-

NAc)-OH (Ic), N^α-Fmoc-Thr(Ac₃-β-D-GlcNAc)-OH (Id),

N^α-Fmoc-Thr(Ac₄-β-D-Gal)-OH (Ie), N^α-Fmoc-Thr(Ac₄-

β-D-Glc)-OH (If) and N^α-Fmoc-Thr(Ac₄-α-D-Man)-OH

(Ig) were synthesized. For the synthesis of Ia and Ib

(Scheme 1), per-O-acetyl 2-azido-2-deoxy-glycopyranosyl

bromides were selected as glycosyl donors containing non-

participating group at C-2. The preparation of these

glycosyl donors involved the azidonitration of galactal

and glucal followed by their conversion to halides [28]. In

case of azidonitration of glucal, the products, the mixture of

2-azido-2-deoxy-gluco and 2-azido-2-deoxy-manno deriva-

tives [29], could not be separated by chromatography and

the mixture was converted to the corresponding bromides

(1b and 1c). The glycosylation of N^α-Fmoc-threonine

benzyl ester [30] with acyl-protected 2-azido glycosyl

bromides (1a, 1b and 1c) afforded the glycosides 2a [31],

2b and 2c. Reductive acetylation of crude 2a by thiolacetic

acid using pyridine as a solvent provided 3a [32], whereas

crude mixture of 2b and 2c resulted the major products 3b

and 3c, respectively, which could be purified by flash

chromatography. Deprotection of benzyl esters of 3a and

3b, by hydrogenation yielded the final products Ia [31] and

Ib, respectively.

The synthesis of N^α-Fmoc-Thr(Ac₃-β-D-GalNAc)-OH

(Ic) and N^α-Fmoc-Thr(Ac₃-β-D-GlcNAc)-OH (Id), as

shown in the Scheme 2, started from 2-amino-2- deoxy-

D-glycosyl hydrochloride. The hydrochlorides (4a and 4b)

were converted into 5, 6, and 7 by well established

Glycoconj J (2011) 28:537–555

539

Fig. 1 Different glycosylated

analogs of Threonine used

for synthesizing differently

glycosylated peptides

OAc

O

AcO

FmocNH

AcNH

O

Ib

CO H

2

Ia

CH

3

CH

3

N^α-Fmoc-Thr(Ac₃-α-D-GalNAc)-OH

N^α-Fmoc-Thr(Ac₃-α-D-GlcNAc)-OH

OAc

AcO

OAc

FmocNH

O

AcO

O

CO H

2

AcNH

CO H

2

CH

3

CH

3

Id

Ic

N^α-Fmoc-Thr(Ac₃-β-D-GlcNAc)-OH

N^α-Fmoc-Thr(Ac₃-β-D-GalNAc)-OH

OAc

FmocNH

O

AcO

O

CO H

2

OAc

CO H

2

OAc

CH

3

CH

3

If

Ie

N^α-Fmoc-Thr(Ac₄-β-D-Gal)-OH

N^α-Fmoc-Thr(Ac₄-β-D-Glc)-OH

OAc

O

AcO

FmocNH

O

CO H

2

Ig

CH

3

N^α-Fmoc-Thr(Ac₄-α-D-Man)-OH

procedures [33]. Reaction of trichloroacetimidates (7a and

7b) with N^α-Fmoc-threonine benzyl ester in the presence

of catalytic amounts of trimethylsilyl trifluoromethanesulfo-

nate (TMSOTf) provided the β-linked glycosides (8a and 8b)

in good yields. Subsequent replacement of the N-

trichloroethoxycarbonyl group in 8 by the N-acetyl group

with zinc and acetic anhydride afforded 9. Finally,

benzyl esters of 9a and 9b were reduced with Pd/C under

H₂atmosphere, which provided the desired products Ic

and Id, respectively.

presence of catalytic amounts of TMSOTf afforded high

yield of β-linked glycosides 14a and 14b, respectively.

Subsequent removal of benzyl esters by hydrogenolysis

yielded the final products Ie and If (Scheme 3).

The classical Koenigs-Knorr method [35] was used for the

synthesis of N^α-Fmoc-Thr(Ac₄-α-D-Man)-OH (Ig). The

synthesis of Ig was achieved by the glycosylation of N^α-

Fmoc-threonine benzyl ester with per-O-acetylated mannosyl

bromide 16 [36], which afforded nearly quantitative yields of

α-glycoside 17. Careful hydrogenolysis of 17 afforded the

desired product Ig (Scheme 4).

The resulting glycosylated amino acid building blocks were

used for synthesizing antibacterial peptides, formaecin I and

M-drosocin, and their various glycosylated analogs (Table 1).

The glycopeptides were synthesized by the solid phase

methodology. Briefly, the peptide chain was first assembled

on the resin by standard Fmoc/DCC-HOBt protocol until the

glycosylated threonine residue position. The glycosylated

threonine was then coupled to the NH₂-peptide resin by

Fmoc/HBTU protocol [37]. Further elongation of the peptide

chain was carried out according to the standard Fmoc/

For synthesizing N^α-Fmoc-Thr(Ac₄-β-D-Gal)-OH (Ie)

and N^α-Fmoc-Thr(Ac₄-β-D-Glc)-OH (If), first the per-O-

acetylated glycopyranoses (11a and 11b) were transformed

with hydrazine acetate into their corresponding 1-O-

unprotected pyranoses (12a and 12b), which were converted

to trichloroacetimidates by treatment with trichloroacetoni-

trile in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene

(DBU) as base following earlier reported protocol [34]. The

glycosylation of N^α-Fmoc-benzyl protected threonine with

the per-O-acetylated galactose trichloroacetimidate (13a) and

per-O-acetylated glucose trichloroacetimidate (13b) in the

540

Glycoconj J (2011) 28:537–555

Scheme 1 Reagents and condi-

tions: (a) Ag₂CO₃, AgClO₄,

Fmoc-Thr-OBn, CH₂Cl₂/

Toluene, 4d; (b) Pyridine,

thiolacetic acid; (c) H₂,

4

OAc

R

OAc

R

2

O

2

O

3

R

a

R

AcO

O

1

R

Br

10%Pd/C, MeOH or EtOAc

FmocHN

Thr

OBn

1

2

3

4

1

2

3

4

1 a R =N , R =H, R =H, R =OAc

3

2 a R =N , R =H, R =H, R =OAc

3

1

2

3

4

1

2

3

4

1 b R =N , R =H, R =OAc, R =H

3

2 b R =N , R =H, R =OAc, R =H

3

1

2

3

4

1

2

3

4

1 c R =H, R =N , R =OAc, R =H

3

2 c R =H, R =N , R =OAc, R =H

3

b

4

OAc

R

2

O

4

OAc

R

3

2

O

R

3

AcO

R

c

1

AcO

R

1

R

O

FmocHN

Thr

OH

FmocHN

Thr

OBn

1

2

3

4

I a R =NHAc, R =H, R =H, R =OAc

1

2

3

4

3 a R =NHAc, R =H, R =H, R =OAc

1

2

3

4

I b R =NHAc, R =H, R =OAc, R =H

1

2

3

4

3 b R =NHAc, R =H, R =OAc, R =H

1

2

3

4

3 c R =H, R =NHAc, R =OAc, R =H

DCC-HOBt protocol. Finally, the deprotection and cleavage

of peptides from resin provided the glycopeptides with acetyl

groups protected sugar. The removal of acetate groups was

achieved by treatment of purified peptide with 5% hydrazine

hydrate. Finally, all the peptides were repurified and charac-

terized by mass spectrometry (Table 1).

Scheme 2 Reagents and condi-

tions: (a) Teoc-Cl, NaHCO₃; (b)

Py, Ac₂O; (c) DMF, N₂H₄

HOAc; (d) CH₂Cl₂, CCl₃CN,

DBU; (e) CH₂Cl₂, Fmoc-Thr-

OBn, TMSOTf; (f) Zn, Ac₂O, rt;

(g) H₂, 10%Pd/C, MeOH

OAc

R

OAc

R

OH

1

c

O

1

R

a, b

O

R

1

R

AcO

OAc

OH

NH HCl

HO

NH

2

O

Cl C

3

1

6 a R=OAc, R =H

4 a R=OH, R =H

4 b R =OH, R=H

1

5 a R=OAc, R =H

5 b R =OAc, R=H

1

6 b R =OAc, R=H

1

d

OAc

FmocNH

R

OAc

O

R

O

1

R

1

e

O

Thr

R

AcO

OBn

AcO

NH

HN

O

NH

O

Cl C

3

O

Cl C

3

CCl

3

1

8 a R=OAc, R =H

1

8 b R =OAc, R=H

1

7 a R=OAc, R =H

7 b R =OAc, R=H

1

f

OAc

O

R

FmocNH

Thr

OAc

FmocNH

1

O

g

R

O

OBn

AcO

O

Thr

AcO

OH

HNAc

1

9 a R=OAc, R =H

I c R=OAc, R =H

1

9 b R =OAc, R=H

I d R =OAc, R=H

Glycoconj J (2011) 28:537–555

541

Scheme 3 Reagents and condi-

tions: (a) Pyridine, Ac₂O; (b)

DMF, N₂H₄HOAc; (c) CH₂Cl₂,

CCl₃CN, DBU; (d) CH₂Cl₂,

Fmoc-Thr-OBn, TMSOTf; (e)

H₂, 10%Pd/C, MeOH or EtOAc

R

OAc

R

OH

R

R¹

HO

a

R¹

AcO

O

R¹

AcO

O

b

O

OH

OAc

OH

OAc

10 a R =OH, R¹=H

10 b R¹=OH, R =H

12 a R =OAc, R¹=H

12 b R¹=OAc, R =H

11 a R =OAc, R¹=H

11 b R¹=OAc, R =H

c

OAc

R

OAc

FmocNH

R¹

d

O

R¹

AcO

O

AcO

O

Thr

OBn

AcO

OAc

O

NH

14 a R =OAc, R¹=H

14 b R¹=OAc, R =H

13 a R =OAc, R¹=H

13 b R¹=OAc, R =H

e

CCl₃

OAc

R

FmocNH

R¹

AcO

O

Thr

O

OH

OAc

I e R =OAc, R¹=H

I f R¹=OAc, R =H

Antibacterial activity

bacterial strains (Figs. 2 and 3). In case of formaecin I, the

change of sugar linkage configuration from α- to β- resulted

in a decrease of 30–40% in antibacterial activity of β-

GalNAc-formaecin I (III) in comparison to that of α-

GalNAc-formaecin I (I) (Fig. 2). With improved antibacterial

activity of the α-GlcNAc-formaecin (IV) in comparison to

that of III, the importance of α linkage is ascertained.

However, IV did not exhibit equivalent levels of antibacterial

activity to native peptide I, suggesting the importance of

configuration of the sugar linkage to peptide as well as axial

orientation of the hydroxyl group at C4. Antibacterial

activity comparison between synthetic α-GlcNAc- (IV) and

β-GlcNAc- (VI) containing formaecin I analogs resulted into

20–35% decrease in the activity of VI as compared to

that of IV, which further emphasizes the importance of

Earlier it has been reported that formaecin I (I) and

monoglycosylated drosocin (M-drosocin) (IX) have higher

antibacterial activities than their non-glycosylated counter-

parts [14, 15, 25]. The synthetic differently glycosylated

peptides of formaecin I and M-drosocin as well as their

respective non-glycosylated analogs were evaluated at

25 nmoles for their antibacterial activities against Gram-

negative bacterial strains of E. coli ATCC 25922, E. coli

BL21λD3, S. typhimurium, and S. typhi Vi⁺. It was

observed that formaecin I (I), M-drosocin (IX) and their

corresponding glycosylated analogs exhibited higher activ-

ities in comparison to their respective non-glycosylated

peptides, II and X, against all the tested Gram-negative

Scheme 4 Reagents and

conditions: (a) Py, Ac₂O; (b)

CH₂Cl₂, 33%HBr in AcOH ; (c)

CH₂Cl₂, Fmoc-Thr-OBn, Mo-

lecular sieves, Silver triflate; (d)

H₂, 5%Pd/C, MeOH

OAc

OH

OAc

O

OAc

O

OH

O

a

b

HO

AcO

OH

OAc

15

16

Br

c

OAc

O

d

AcO

17

O

Thr

I g

FmocHN

O

FmocHN

OBn

Thr

OH

542

Glycoconj J (2011) 28:537–555

Table 1 Amino acid sequences of formaecin I, M-drosocin, their non-glycosylated and different sugar containing analogs

Peptide

Sequence

MALDI-

TOF MS

[M+H]

MALDI-

TOF MS

[M+H]

Calcd

Exptl

1997.0691

1997.0752

OH

Formaecin I

O

(I)

HO

AcHN

O

GRPNPVNNKPTPHPRL

1793.9897^a1793.2686

Nonglycosylated

formaecin I

(II)

1997.0691

1997.0917

1997.0968

OH

-GalNAc-

formaecin I

(III)

O

HO

AcHN

GRPNPVNNKPTPHPRL

OH

O

-GlcNAc-

formaecin

(IV)

HO

AcHN

O

GRPNPVNNKPTPHPRL

1956.0425

1997.0691

1956.0793

1997.0826

OH

-Man-

formaecin I

(V)

O

HO

O

GRPNPVNNKPTPHPRL

-GlcNAc-

formaecin I

(VI)

OH

O

HO

O

AcHN

GRPNPVNNKPTPHPRL

1956.0425

1956.0541

1956.0729

-Gal-

formaecin I

(VII)

HO

OH

O

HO

OH

GRPNPVNNKPTPHPRL

-Glc-

OH

formaecin I

(VIII)

O

HO

O

HO

OH

GRPNPVNNKPTPHPRL

α- configuration of the sugar linkage. The replacement of

α-GalNAc- in formaecin I (I) with α-Man- (V) resulted in

decrease in its antibacterial activity level up to 50%,

probably because of stereochemistry at both C2 and C4

positions is different to that of α-GalNAc- present in

native formaecin I. To examine the influence of equatorial

Glycoconj J (2011) 28:537–555

Table 1 (continued)

543

2401.3230

2401.3933

M-drosocin

OH

(IX)

O

HO

AcHN

O

GKPRPYSPRPTSHPRPIRV

2198.2436^a2198.4484

Nonglycosylated

drosocin

(X)

2401.3230

2401.3347

2401.3765

-GalNAc-

drosocin

(XI)

OH

O

HO

AcHN

GKPRPYSPRPTSHPRPIRV

-GlcNAc-

drosocin

(XII)

OH

O

HO

AcHN

O

GKPRPYSPRPTSHPRPIRV

2360.3766

2360.3169

-Man-

drosocin

(XIII)

OH

O

HO

O

GKPRPYSPRPTSHPRPIRV

2401.3230

2360.3766

2401.3406

2360.3164

2360.3701

OH

-GlcNAc-

drosocin

(XIV)

O

HO

O

AcHN

GKPRPYSPRPTSHPRPIRV

OH

-Gal-

drosocin

(XV)

O

HO

OH

GKPRPYSPRPTSHPRPIRV

OH

-Glc-

drosocin

(XVI)

O

HO

O

OH

GKPRPYSPRPTSHPRPIRV

a

calcd and exptl are abbreviations used for calculated and experimental value respectively, ESI HRMS

orientation of hydroxyl group at C4, comparison in

antibacterial activities was done between α-GalNAc- (I),

α-GlcNAc- (IV) and β-GalNAc- (III), β-GlcNAc- (VI)

containing formaecin analogs. The α-GlcNAc- containing

formaecin analog (IV) showed 10–20% decrease in the

antibacterial activity in comparison to that of formaecin (I)

while β-GlcNAc- (VI) showed 10% lower activity than

III, which supports the fact that axial configuration of the

544

Glycoconj J (2011) 28:537–555

Fig. 2 Antibacterial activity

data of the differently glycosy-

lated analogs of formaecin I

against E.coli ATCC25922 (a),

E. coli BL21λD3 (b),

S. typhimurium (c), and S. typhi

Vi⁺(d), compared with those

of the native formaecin I

b

a

200

160

120

80

200

160

120

80

_β-GaE_lN._Accoli BL21λD3

E. Coli ATCC 25922

β-GalNAc

40

0

[GRPNPVNNKPT(α-GalNAc)

PHPRL] and its non-

glycosylated form

(GRPNPVNNKPTPHPRL) at

25 nmoles. The results represent

the mean S.E.M. of at least

three independent experiments

performed in triplicate

Peptides (25 nmoles)

c

d

200

160

120

80

160

120

80

40

0

S. typhi Vi⁺

S. typhimurium

40

0

Peptides (25 nmoles)

hydroxyl group at C4 plays an important role. Also,

comparative account of antibacterial activity among β-

Gal- (VII) and β-Glc- (VIII) containing formaecin

analogs showed that VII exhibited 20–40% higher activity

against all the tested strains as compared to that of VIII

further suggesting the importance of stereochemistry of

the OH group at C4 position. To probe the replacement of

acetylated amide with OH group at C2, comparison

between β-Gal- (VII) and β-GalNAc- (III) containing

formaecin analogs was done. The β-Gal formaecin

analog (VII) was found to have 5–15% higher antibac-

terial activity than that of β-GalNAc-formaecin analog

Fig. 3 Comparison of antibac-

terial activity of differently

glycosylated analogs of

M-drosocin with M-drosocin

[GKPRPYSPRPT(α-GalNAc)

SHPRPIRV] and its

a

b

400

350

300

250

200

150

E. coli ATCC25922

E. coli BL21λD3

350

300

250

200

150

100

50

100

50

0

non-glycosylated form

(GKPRPYSPRPTSHPRPIRV)

at 25 nmoles against E.coli

ATCC25922 (a), E. coli

BL21λD3 (b), S. typhimurium

(c), and S. typhi Vi⁺(d).

The results represent the

mean S.E.M. of at least three

independent experiments

performed in triplicate

0

Peptides (25 nmoles)

c

d

350

300

250

200

150

100

50

400

350

300

250

200

150

100

50

S. typhi Vi⁺

S. typhimurium

0

Peptides (25 nmoles)

Glycoconj J (2011) 28:537–555

545

(III) giving the assessment of the effect of acetamido

group in III.

drosocin (XV) and β-GalNAc-drosocin (XI) exhibited

15% more activity in β-Gal-drosocin than β-GalNAc-

drosocin (XI).

The antibacterial activity levels of synthetic M-drosocin

(IX) and its analogs (X-XVI) were compared using above

Gram-negative bacterial strains (Fig. 3). It was observed

that the change of configuration of sugar linkage from

α-GalNAc- (IX) to β-GalNAc- (XI) in M-drosocin

resulted in 10–25% decrease in its antibacterial activity.

Also, comparison between α-GlcNAc-drosocin (XII) and

β-GlcNAc-drosocin (XIV) exhibited 10–20% decrease in

the antibacterial activity of XIV to that of XII. The 5–30%

lower levels of activity of α-Man-drosocin (XV) as

compared to IX, when tested against various Gram-

negative strains, showed the influence of stereochemistry

at C2 and C4 hydroxyl groups of sugar on its antibacterial

activity. To assess the importance of axial orientation of

hydroxyl group at C4, the antibacterial activities of α-

GalNAc-drosocin (IX) and β-GalNAc-drosocin (XI) were

compared with α-GlcNAc-drosocin (XII) and β-GlcNAc-

drosocin (XIV), respectively. The activity levels of XII were

found to be 10% lower than that of α-GalNAc-drosocin (IX)

whereas 10–15% decrease in antibacterial activity was

observed for drosocin analog containing β-GlcNAc- (XIV)

as compared to that of β-GalNAc-drosocin (XI). Also, the

equatorial orientation of the OH- group at C4 in β-Glc-

drosocin (XVI) resulted into 10–20% decrease in its

antibacterial activity in comparison to that of β-Gal-

drosocin (XV). To assess the effect of the acetamido

group, the comparison of antibacterial activity of β-Gal-

Though native formaecin I, M-drosocin and their all

glycosylated analogs showed higher antibacterial activities

as compared to their respective non-glycosylated counter-

parts, they showed significant differences among each

other. The pattern of antibacterial activities of different

glycosylated analogs of drosocin (XI–XVI) was comparable

to that of formaecin analogs (III–VIII) but the extent of

decrease in the antibacterial activity of glycosylated drosocin

analogs as compare to that of M-drosocin was less.

Thus, overall comparison of the antibacterial activities of

formaecin I, M-drosocin and their analogs against Gram-

negative bacterial strains showed the following order : α-

GalNAc- > α-GlcNAc- > β-Gal- > β-GalNAc- > β-

GlcNAc- ≈ β-Glc- ≈ α-Man- > non-glycosylated- with the

difference much more pronounced in different glycosylated

analogs of formaecin I than of M-drosocin.

Conformational studies

The comparative conformational properties of formaecin I,

M-drosocin and their glycosylated analogs were analyzed

by CD spectroscopy in water, sodium-dodecyl-sulfate

(10 mM SDS), and 2,2,2- trifluoroethanol (90% TFE).

The CD spectra of formaecin I, M-drosocin and their

glycosylated analogs in water are shown in Figs. 4a and 5a.

The overall spectral profiles for all the glycosylated analogs

Formaecin I (α-GalNAc-formaecin)

β-GalNAc-formaecin

α GlcNAc-formaecin

α Man-formaecin

β GlcNAc-formaecin

β-Gal-formaecin

β Glc-formaecin

b

a

c

50x10⁴

40x10⁴

50x10⁴

0

In 10mM SDS

In 90% TFE

In water

0

-50x10⁴

-1x10⁶

-50x10⁴

-1x10⁶

-1.7x10⁶

-1.3x10⁶

-1.5x10⁶

190 200

220

240 250

190 200

220

240 250

190 200

220

240 250

λ

Fig. 4 CD spectra of the differently glycosylated analogs of

formaecin I in water (a), in 90% TFE (b), and in 10 mM SDS (c)

compared with native formaecin I [GRPNPVNNKPT(α-GalNAc)

PHPRL], suggesting that the consequences of the sugar variation are

not distinguishable by circular dichroism studies

546

Glycoconj J (2011) 28:537–555

M-drosocin ( α-GalNAc -drosocin)

β-GalNAc -drosocin

α GlcNAc -drosocin

α Man-drosocin

β GlcNAc -drosocin

β-Gal-drosocin

β Glc-drosocin

c

a

b

30x10 ⁴

10x10 ⁴

0

In 10mM SDS

In water

0

In 90% TFE

0

-50x10 ⁴

-1x10 ⁶

-50x10 ⁴

-1x10 ⁶

-2x10 ⁶

6

-2.4x10

-1.3x10

-1.5x10

190 200

220

240 250

190 200

220

240 250

190 200

220

240 250

λ

Fig. 5 Circular dichroism profiles of M-drosocin [GKPRPYSPRPT(α-GalNAc)SHPRPIRV] and its differently glycosylated analogs in water (a),

in 90% TFE (b), and in 10 mM SDS (c). All the glycopeptides show similar pattern in different environments

of formaecin and M-drosocin were generally similar

whereas the absolute values of the molar ellipticities varied

slightly among the analogs. The CD spectra of all the

glycopeptides showed a minima around 200 nm and a

negative shoulder at around 235 nm, which are the

characteristics of a disordered structure. All the glycosy-

lated peptides displayed basically random conformations in

water. In lipophilic solvent (90% TFE), all the peptides

exhibited spectral alterations (Figs. 4b and 5b). Formaecin

I, M-drosocin and their glycosylated analogs showed

characteristic two negative bands at about 203 nm and

225 nm and a positive band around 190 nm, similar to the

pattern, which has been assigned to the presence of type-I

(III) β-turns or β-turn mixtures [38, 39] with significant

type-I character also reported for drosocin and its

deglycosylated form [26]. In micellar SDS environment

(Figs. 4c and 5c), all the glycopeptides exhibited similar

CD spectra which were characterized by a broad negative

band at around 202 nm somewhat red shifted and less

intense than that observed in water, suggesting a minor

stabilization of the turn structure without clear effect on

the peptide conformation.

Cytotoxicity of glycosylated peptides

In vitro cytotoxic assays are useful to evaluate the intrinsic

ability of the peptide to cause cell death as a consequence

of interference in several cellular functions. To investigate

the in vitro cytotoxicity of these antimicrobial peptides on

eukaryotic cells, the hemolytic activity and MTT assays

were done. The effects of formaecin I, M-drosocin, their

differently glycosylated analogs and respective non-

glycosylated analogs on murine erythrocytes and SP2/O

cell lines were evaluated.

The hemolytic activities of formaecin I, M-drosocin and

their respective analogs II–VIII and X–XVI were deter-

mined as the percentage of lysis of rat erythrocytes. It was

observed that formaecin I, its non-glycosylated form (II)

and glycosylated analogs (III–VIII) showed less than 5%

hemolytic activity at the concentration of 100 μM, when

tested on 2% (vol/vol) suspensions of rat erythrocytes. All

these peptides were non-toxic to rat blood cells. Similar

results were obtained with M-drosocin and its analogs X–

XVI, when tested for their hemolytic activities on rat

erythrocytes at a concentration of 100 μM used. Thus,

hemolytic activity was absent in formaecin I, M-drosocin

and in their respective non-glycosylated peptides as well as

their differently glycosylated analogs (Fig. 6a and b).

The cytotoxic potential of all the synthesized peptides was

also determined by following incubation of exponentially

growing SP2/O cells with these peptides using MTTassay. No

difference was found between the cells in the control group

and the cells incubated with formaecin I, M-drosocin and their

Thus, conformational studies by CD of formaecin I,

M-drosocin and their glycosylated analogs did not show

any peculiar difference among each other. In general

they displayed random conformations in water. In the

membrane mimicking environments like TFE and SDS,

all these peptides could not acquire any appreciable

ordered structure except for the presence of β-turns as

exhibited by their CD spectra.

Glycoconj J (2011) 28:537–555

547

a

b

120

100

80

60

40

20

0

100

80

60

40

20

0

Peptides [100μM]

Fig. 6 Hemolytic potential of formaecin I [GRPNPVNNKPT(α-

GalNAc)PHPRL], its non-glycosylated (GRPNPVNNKPTPHPRL)

and differently glycosylated analogs (a), and M-drosocin

[GKPRPYSPRPT(α-GalNAc)SHPRPIRV], its non-glycosylated

(GKPRPYSPRPTSHPRPIRV) and various glycosylated analogs (b)

at concentration of 100 μM, on rat erythrocytes following 1 h

exposure. The results represent the mean S.E.M. of at least three

independent experiments performed in duplicate

non-glycosylated and glycosylated analogs for 24 h (Fig. 7a

and b). All the tested peptides caused no decrease in cell

viability even with the concentration of 50 mg/ml. Thus, all

the tested glycosylated peptides did not show cytotoxicity on

SP2/O cells in the present study.

the useful model system for studying structural and

physiological consequences of protein glycosylation.

The proline-rich class of antibacterial glycopeptides such

as formaecins, drosocins, pyrrhocoricin, diptericin and

lebocins from insects contain conserved glycosylated

threonine residue [14, 15]. The integrity of sugar moieties

is necessary for maximum activity of most of these

antimicrobial peptides. We have synthesized a variety of

O-linked glycosylated threonine residues carrying common

sugar moieties present in eukaryotic proteoglycans with

variable anomeric configuration and incorporated into two

naturally occurring glycosylated antimicrobial peptides,

formaecin I and M-Drosocin, and studied their functional

implications.

Discussion

The importance of protein glycosylation in a variety of

biological processes is known. However, the specific contri-

bution of carbohydrate moieties often remains elusive. The

issues related to biological function of glycoconjugates can be

evaluated, provided suitable quantities of rational model

systems can be made available. The chemical syntheses of

homogenous glycopeptides have allowed generating a diverse

array of carbohydrate structures for studying their

functions and local perturbations in a systematic

fashion. The synthetic antibacterial glycopeptides are

A variety of glycopeptides have been studied to elucidate

the impact of N- linked or O-linked sugar chains on their

function as well as on the conformation and flexibility of

peptides by NMR [8, 10, 13, 40, 41]. It has been shown that

O-linked carbohydrates alter the peptide-core conformation

a

b

140

120

100

80

60

40

20

0

120

100

80

60

40

20

0

Peptides [50mg/ml]

Fig. 7 Effect of formaecin I [GRPNPVNNKPT(α-GalNAc)PHPRL],

its non-glycosylated (GRPNPVNNKPTPHPRL) and differently gly-

cosylated analogs (a), and M-drosocin [GKPRPYSPRPT(α-GalNAc)

SHPRPIRV], its non-glycosylated (GKPRPYSPRPTSHPRPIRV) and

various glycosylated analogs (b) at concentration of 50 mg/ml on the

viability of SP2/O cells following 24 h incubation at 37°C using the

MTT assay. The results represent the mean S.E.M. of at least three

independent experiments performed in duplicate

548

Glycoconj J (2011) 28:537–555

and reduce the local mobility around the glycosylation site

[10, 41–43]. In case of drosocin, the comparative NMR

studies [24] of glycosylated and non-glycosylated forms

suggest that glycosylation provides a more extended

conformation around the glycosylation site and modifies

turns at other parts of the molecule. However, these

structural differences were not evident with CD analysis

as circular dichroism spectra gave information of overall

structure, not of local elements, which were random in both

cases. But the differences in antibacterial activities between

glycosylated and non-glycosylated drosocin were signifi-

cant. In the present analysis, CD spectra of differently

glycosylated peptides reveal no evidence of regular sec-

ondary structures in different solvents and are therefore not

distinguishable. However, all the glycosylated analogs of

formaecin I and drosocin exhibit noticeable differences in

antibacterial activities among each other despite having the

same peptide sequence. This variation may be due to

dissimilarities in the conformations induced by different

sugar moieties among the individual glycopeptide and/or

topological differences among sugars influencing binding

to yet unknown receptor/receptors. We also observed that

magnitude of variation in antibacterial activity was much

more pronounced in formaecin analogs than in glycosylated

analogs of M-drosocin. Previous findings indicate that

conformational change of glycosylation also depends on

the context of peptide sequence [44]. So, the difference in

the variation of antibacterial activities between formaecin I

glycosylated analogs and M-drosocin glycosylated analogs,

can be best explained by sequence-specific glycosylation

induced conformational change. All the antibacterial

glycopeptides studied here show higher antibacterial

activities as compared to their non-glycosylated counterparts.

It appears that sugar conjugation provides higher

conformational stability as compared to non-glycosylated

analogs [8, 10, 13, 40, 41, 45].

In our study we found the attribute of carbohydrate

attachment to peptide (via α- or β-glycosidic bond) within

a locus has a contemplative effect on the antibacterial

activity against Gram-negative bacteria. β-linked deriva-

tives in both formaecin I and M-drosocin show significant

differences in their antibacterial activities as compared to α-

linked glycopeptides of formaecin I and M-drosocin,

respectively. In general, the pattern of β-linked sugar in

the simplified cases [44, 46, 47] exhibits considerable

flexibility in the peptide backbone with dynamic and

relatively unstrained conformations unlike their α-linked

analogs, which are confined to limited conformations with

remarkable stability and extended structures. The observed

reduction in activity by changing the anomeric config-

uration of sugar from α- to β- in case of formaecin I

and M-drosocin glycosylated analogs is in accordance

with the work of Coltart et al. [46] and Wu et al. [44].

All β-derivatives studied here show activities lower than

those of the corresponding α-derivatives.

The antibacterial activity comparison among the glyco-

peptides with α-GalNAc, α-GlcNAc and β-GalNAc, β-

GlcNAc and β-Gal, β-Glc exhibited slightly higher activity

of peptide derivatives linked with GalNAc/Gal moiety than

GlcNAc/Glc moiety. This variation in activity can be

attributed to the difference in the stereochemistry at C-4

with axial and equatorial OH-group in GalNAc/Gal and

GlcNAc/Glc, respectively. The comparative antibacterial

activity profile of β-Gal and β-GalNAc containing analogs

reflected the slightly higher activity of β-Gal derivative than

that of β-GalNAc derivative. The approach and participation

of acetamide protons of β-GalNAc causing hindrance in

binding to the target molecule might be an explanation of

comparatively higher activity of β-Gal-derivative.

Effect of glycosylation seems to be dependent on

configuration of the sugar linkage as well as nature and

stereochemistry of substituents at the C-2 and C-4 position

and in the milieu of amino acid residues of peptides as well.

Change in conformational stability and/or modulation in

binding properties due to stereochemical differences at C-2

and C-4 can be best possibly assigned for the minimum

activity of a mannose containing analog. Thus, our analysis

indicates that antibacterial activity of formaecin I and M-

drosocin against Gram-negative strains studied here can be

achieved maximally by glycosylation with a GalNAc sugar

moiety appended in α-glycosidic linkage to threonine

residue. The antibacterial activity determination of various

glycosylated analogs in relation to two different peptide

sequences provides a good model system to study

functional consequences of sugar variants.

The comparative analyses of cell cytotoxicity examined by

hemolytic activity and MTT assay for formaecin I, M-

drosocin and their glycosylated analogs showed no surprising

results by change of sugar as both native and their differently

glycosylated derivatives were found to be devoid of any kind

of toxicity.

Conclusions

The synthesis of O-linked antibacterial glycopeptides,

formaecin I and M-drosocin, with varied carbohydrate

moieties permitted access to structurally diverse molecules,

which are explored for the resulting impact on their

antibacterial activity. All the differently glycosylated

analogs were found to be more potent than their non-

glycosylated counterpart. We have shown that the glycosy-

lated peptides differing in nature of sugar and its linkage to

the peptide of same amino acid sequences exhibited

different antibacterial activities most likely due to differen-

tial topological effect of glycosylation in particular and

Glycoconj J (2011) 28:537–555

549

peptide conformation in general. Indeed, these effects are

found to be sequence-specific and therefore, also depend on

the context of peptide sequence. The high-resolution

conformational analyses of these glycopeptides are required

to address the precise effect of different O-linked sugars.

The cytotoxicity experiments showed that native antibacte-

rial peptides or their designed analogs are non-hemolytic

and non-toxic to eukaryotic cells. Our findings will be

useful for understanding the effect of sugar variants in

glycopeptide antibiotics and designing of glycosylated

antibacterial agents.

product was purified by flash chromatography using

(gradient of 0–60% EtOAc-Hexane over 40 min). The

appropriate fractions were combined and concentrated to

give desired product 3a (2.28 g, 65%). ¹H NMR (300 MHz,

CDCl₃): δ 7.77 (d, J=7.2 Hz, 2H, Ar), 7.63 (d, J=7.2 Hz,

2H, Ar), 7.43–7.30 (m, 9H, Ar), 5.81 (d, J=9.9 Hz, 1H,

NH), 5.67 (d, J=9.3 Hz, 1H, NH), 5.37 (br s, 1H, H-4),

5.21–5.05 (m, 3H, H-3, OCH₂Ph), 4.79 (d, J=3.3 Hz, 1H,

H-1), 4.56–4.43 (m, 4H, β-H, FmocCH₂, α-H), 4.28–4.05

(m, 5H, FmocCH, H-6ª^,b, H-2, H-5), 2.16, 2.02, 2.00, 1.96

(4 s, each 3H, 4Ac), 1.29 (d, J=6.0 Hz, 3H, Thr-CH₃). All

data are in agreement with literature reports [32]. ESI

HRMS: calcd for C₄₀H₄₄N₂O₁₃Na[M+Na]⁺m/z, 783.2741;

found, 783.5074.

Experimental section

Material and methods HMP (4-hydroxymethyl phenoxy-

methyl polystyrene) resin, solvents and reagents used for

synthesis were supplied by Applied Biosystems Inc. Fmoc

amino acid derivatives were procured from Bachem

Feinchemikalein AG. Agrose I (Biotechnology grade) was

obtained from Amresco, and tryptic soy broth (TSB) was

from Himedia Laboratories Pvt. Ltd. All reactions sensitive

to air and/or moisture were carried out under argon

atmosphere with anhydrous solvents. NMR spectra were

recorded on a BRUKER 300 Ultrashield™ NMR

spectrometer. Molecular mass determinations were made

by using ESI-MS system (Applied Biosystems Mariner

System 5220) operating in positive mode. Chromatographic

separations were performed on a silica gel column by flash

chromatography (Combi Flash Companion, Teledyne ISCO).

Yields are given after purification, unless differently stated.

N^α-fluoren-9-ylmethoxycarbony-O-(2-acetamido-2-deoxy-

3,4,6-tri-O-acetyl-α-D-galactopyranosyl)-L-threonine (Ia)

To a solution of 3a (982 mg, 1.29 mmol) in methanol

(30 ml) was added 10% Pd/C (390 mg). The mixture was

stirred under one atmosphere of hydrogen gas for 2 h. The

catalyst was removed by filtration and after solvent

evaporation, the residue was purified by flash chromatog-

raphy using CHCl₃as solvent A and 50% MeOH-CHCl₃as

solvent B (gradient of 0–30% B over 35 min) to give the

1

titled compound Ia (600 mg, 69%). H NMR (300 MHz,

CD₃OD): δ 7.82 (d, J=7.2 Hz, 2H, Ar), 7.71–7.67 (m, 2H,

Ar), 7.42–7.29 (m, 4H, Ar), 5.39 (app d, J=3.0 Hz, 1H,

H-4), 5.07 (dd, J=3.2 Hz, J=11.5 Hz, 1H, H-3), 4.94 (d,

J=3.8 Hz, 1H, H-1), 4.62–4.35 (m, 4H, FmocCH₂, β-H,

H-6ª), 4.31–4.23 (m, 3H, FmocCH, α-H, H-6^b), 4.14–4.05

(m, 2H, H-5, H-2), 2.14, 2.03, 1.94, 1.93 (4 s, each 3H,

4Ac), 1.24 (d, J=6.4 Hz, 3H, Thr-CH₃); The ¹H NMR data

is in accord with the literature [31]. ¹³C NMR (75 MHz,

CD₃OD): δ 172.3, 170.8, 170.7, 170.6 (COCH₃), 157.7

(CO Fmoc), 144.0, 143.7, 141.3, 127.4, 126.8, 124.8,

124.6, 119.6, 119.5 (Ar C), 99.3 (C-1), 76.3 (β-C), 68.3 (C-3),

67.4 (C-4), 66.8, 66.3, 61.9, 58.6 (CH₂Fmoc, C-6, α-C, C-5,

C-2), 47.1 (CH Fmoc), 21.5, 19.3, 19.2, 19.1 (COCH₃), 17.8

(CH₃Thr). ESI HRMS: calcd for C₃₃H₃₈N₂O₁₃Na[M+Na]⁺

m/z, 693.2271, found, 693.3944.

N^α-fluoren-9-ylmethoxycarbonyl-O-(2-acetamido-2-deoxy-

3,4,6-tri-O-acetyl-α-D-galactopyranosyl)-L-threonine

benzyl ester (3a) To a solution of N-Fmoc-L-threonine

benzyl ester (2 g, 4.63 mmol) in dry CH₂Cl₂-Toluene(1:1,

20 ml) was added 4A° molecular sieves (3 g) and Ag₂CO₃

(3.83 g, 13.9 mmol) and stirred at room temperature for

45 min. AgClO₄(1.2 g, 5.78 mmol) was added and stirred

for another 20 min. A solution of azidobromide 1a (3.28 g,

8.33 mmol) in dry CH₂Cl₂-Toluene(1:1) was slowly added

and the reaction mixture was stirred at room temperature for

4 days. The reaction mixture was diluted with CH₂Cl₂and

filtered over celite. The celite was rinsed with CH₂Cl₂

thoroughly until no product was retained. The filtrate was

washed with saturated NaHCO₃and H₂O. All the aqueous

washes were back extracted with CH₂Cl₂. The pooled

organic layers were dried over Na₂SO₄and concentrated to

get the product 2a (crude 3.38 g). To a solution of 2a

(3.38 g) in pyridine (35 ml), thiolacetic acid (35 ml) was

added. The reaction mixture was allowed to stir for 4 h at

room temperature. The reaction mixture was diluted with

toluene and concentrated under reduced pressure. The crude

N^α-fluoren-9-ylmethoxycarbonyl-O-(2-acetamido-2-deoxy-

3,4,6-tri-O-acetyl-α-D-glucopyranosyl)-L-threonine benzyl

ester (3b) A mixture of 1b and 1c (2.16 g, 5.48 mmol) and

Fmoc-Thr-OBn (1.3 g, 3.01 mmol), was treated as

described for the synthesis of 2a. The crude mixture of

azido-glycosides (2b and 2c) was taken directly for the next

reaction. The mixture (2.1 g) was dissolved in pyridine

(30 ml) and thiolacetic acid (30 ml) was added to it. The

mixture was stirred at room temperature for overnight. The

reaction mixture was diluted with toluene and concentrated

under reduced pressure. The residue was purified by flash

chromatography using (gradient of 0–60% EtOAc-Hexane

550

Glycoconj J (2011) 28:537–555

over 40 min) to give two major compounds, 3b (880 mg,

38%) and 3c (800 mg, 35%). The ¹H NMR confirmed 3b to

be the title compound and 3c was characterized as N^α-

fluoren-9-ylmethoxycarbonyl-O-(2-acetamido-2-deoxy-

3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-L-threonine ben-

(d, J=3.8 Hz, H-1), 4.92 (d, J=3.3 Hz, H-1), 4.74–4.61

(m), 4.55–4.44 (m), 4.34–4.18 (m), 4.13–3.98 (m), 3.92–

3.81 (m), 2.09 (s, Ac), 2.08 (s, Ac), 2.06 (s, Ac), 2.01(s,

Ac), 2.00 (s, Ac), 1.96 (s, Ac), 1.90 (s, Ac), 1.26 (d, J=

5.9 Hz, Thr-CH₃), 0.91 (d, J=6.2 Hz, Thr-CH₃). ¹³C NMR

(75 MHz, CDCl₃) δ 99.3 (C-1), 98.3 (C-1). ¹H NMR

(75 MHz, CD₃OD) δ7.82 (d, J=7.4 Hz, 2H, Ar), 7.70 (app

t, J=7.0 Hz, 2H, Ar), 7.43–7.29 (m, 4H, Ar), 5.20 (dd, J=

9.5 Hz, J=10.7 Hz, 1H, H-3), 4.97 (t, J=9.7 Hz, 1H, H-4),

4.89 (H-1 overlap with HOD), 4.63–4.39 (m, 3H, Fmoc-

CH₂, β-H), 4.29–4.05 (m, 6H, α-H, FmocCH, H-6ª^,b, H-2,

H-5), 2.05, 2.04, 1.98, 1.92 (4 s, each 3H, 4Ac), 1.22 (d, J=

6.4 Hz, 3H, Thr-CH₃). ¹³C NMR (75 MHz, CD₃OD) δ

172.0, 171.0, 170.8, 169.9 (COCH₃), 157.8 (CO Fmoc),

144.0, 143.8, 141.3, 141.3, 127.4, 126.8, 124.8, 124.7,

119.6, 119.5 (Ar C), 98.7 (C-1), 76.4 (β-C), 71.0, 68.9,

67.9 (C-3, C-4, C-5), 66.3 (CH2 Fmoc), 62.1 (C-6), 58.3

(α-C), 51.3 (C-2), 46.8 (CH Fmoc), 21.4, 19.2 (COCH₃),

17.8 (CH₃Thr). ESI HRMS: calcd for C₃₃H₃₉N₂O₁₃[M+H]⁺

m/z, 671.2452; found, 671.4193.

1

zyl ester. 3b H NMR (300 MHz, CDCl₃): δ 7.79 (d, J=

7.4 Hz, 2H, Ar), 7.66 (d, J=7.2 Hz, 2H, Ar), 7.44–7.30

(m, 9H, Ar), 5.94 (d, J=9.5 Hz, 1H, NH), 5.72 (d, J=

9.4 Hz 1H, NH), 5.21–5.05 (m, 4H, H-3, H-4, OCH₂Ph),

4.71 (d, J=3.6 Hz, 1H, H-1), 4.50–4.42 (m, 3H, α-H,

FmocCH₂), 4.33–4.06 (m, 5H, H-6ª^,b, β-H, FmocCH,

H-2), 4.02–3.97 (m, 1H, H-5), 2.08, 2.05, 2.04, 1.97 (4 s,

each 3H, 4Ac), 1.30 (d, J=6.3 Hz, 3H, Thr-CH₃); ¹³C

NMR (75 MHz, CDCl₃): δ 171.3, 170.7, 170.6, 170.3

(COCH3), 169.2, 156.5 (CO Fmoc), 143.8, 143.7, 141.3,

134.4, 129.0, 128.9, 128.6, 127.8, 127.1, 125.1, 120.0

(Ar C), 99.3 (C-1), 77.3 (β-C), 71.2 (C-3), 68.3, 67.8,

67.4 (C-4, C-5, OCH₂Ph, CH₂Fmoc), 62.1, 58.6, 51.6 (C-6,

α-C, C-2), 47.2 (CH Fmoc), 23.0, 20.7, 20.6 (COCH3), 18.3

(CH₃Thr). ESI HRMS: calcd for C₄₀H₄₅N₂O₁₃[M+H]⁺

1

m/z: 761.2933; found, 761.4794. 3c H NMR (300 MHz,

CDCl₃): δ7.70 (d, J=7.4 Hz, 2H, Ar), 7.57 (d=7.3 Hz,

2H, Ar), 7.36–7.23 (m, 9H, Ar), 5.67 (d, J=9.4 Hz, 1H,

NH), 5.65 (d, J=8.4 Hz, 1H, NH), 5.24–5.15 (m, 3H, H-3,

OCH₂Ph), 4.98 (t, J=9.9 Hz, 1H, H-4), 4.73 (br s, 1H, H-1),

4.46–4.16 (m, 6H, β-H, FmocCH₂, α-H, FmocCH, H-6ª),

4.08–3.97 (m, 3H, H-6^b, H-5, H-2), 2.01, 1.99, 1.97, 1.94

(4 s, each 3H, 4Ac), 1.24 (d, J=6.3 Hz, 3H, Thr-CH₃). ¹³C

NMR (CDCl₃): δ 170.4, 169.9, 169.8 (COCH₃), 156.6 (CO

Fmoc), 143.8, 143.7, 141.2, 135.1, 128.6, 128.5, 128.4,

127.6, 127.1, 127.0, 125.2, 119.9 (Ar C), 100.1 (C-1), 77.2

(β-C), 68.8 (C-3), 68.6, 67.8, 67.5, 66.2, 62.5, 58.6, 50.6

(OCH₂Ph, C-4, C-5, α-C, CH₂Fmoc, C-6, C-2), 47.1 (CH

Fmoc), 23.3, 20.7, 20.6 (COCH₃), 17.8 (CH₃Thr). ESI

HRMS: calcd for C₄₀H₄₄N₂O₁₃Na [M+Na]⁺m/z, 783.2741,

found, 783.4616.

N^α-fluoren-9-ylmethoxycarbonyl-O-[3,4,6-Tri-O-acetyl-2-

deoxy-2-(2,2,2-trichloro- ethoxy-carbonylamino)-β-D-

galactopyranosyl]-L-threonine benzyl ester (8a) A reac-

tion mixture of Fmoc-Thr-OBn (1.7 g, 3.93 mmol),

trichloroacetimidate 7a (3.68 g, 5.895 mmol) and activated

4A° molecular sieves in dry CH₂Cl₂was stirred at room

temperature for 30 min. under argon. TMSOTf (11 μl,

0.06 mmol) was added at 0°C. The reaction mixture was

warmed to room temperature and stirred until completion

indicated by TLC. The mixture was diluted with CH₂Cl₂

and quenched with a few drops of triethylamine. The

solution was filtered, washed with saturated NaHCO₃, H₂O

and dried over MgSO4. Purification by flash chromatography

(gradient of 0–40% EtOAc-Hexane over 35 min) afforded 8a

(2.62 g, 75%) as a white solid. ¹H NMR (300 MHz, CDCl₃)

δ 7.76 (d, J=7.5 Hz, 2H, Ar), 7.65 (m, 2H, Ar), 7.42–7.29

(m, 9H, Ar), 5.83 (d, J=9.0 Hz, 1H, NH), 5.32 (d, J=2.8 Hz,

1H, H-4), 5.23–5.09 (m, 4H, H-3, NH, OCH₂Ph), 4.78–4.65

(m, 2H, TrocCH₂), 4.56–4.42 (m, 4H, FmocCH₂, H-1, β-H),

4.33–4.23 (m, 2H, α-H, FmocCH), 4.14–3.99 (m, 2H,

H-6ª^,b), 3.81–3.69 (m, 2H, H-5, H-2), 2.14, 2.04, 2.02

(3 s, each 3H, 3Ac), 1.24 (d, J=6.3 Hz, 3H, Thr-CH₃). ESI

HRMS: calcd for C₄₁H₄₄N₂O₁₄Cl₃[M+H]⁺m/z,

893.1860; found, 893.3827.

N^α-fluoren-9-ylmethoxycarbonyl-O-(2-acetamido-2-deoxy-

3,4,6-tri-O-acetyl-α-D-glucopyranosyl)-L-threonine (Ib)

To a solution of 3b (680 mg, 0.89 mmol) in ethyl acetate

(20 ml) was added 10% Pd/C (340 mg). The mixture was

stirred under one atmosphere of hydrogen gas until the

reaction was complete. The catalyst was removed by

filtration and after solvent evaporation, the residue was

purified by flash chromatography using DCM as solvent A

and 10% MeOH-DCM as solvent B (gradient of 0–50% B

1

over 40 min) to afford pure Ib (396 mg, 66%). The H-

N^α-fluoren-9-ylmethoxycarbonyl-O-(2-acetamido-2-deoxy-

3,4,6-tri-O-acetyl-β-D-galactopyranosyl)-L-threonine ben-

zyl ester (9a) To the solution of 8a (2 g, 2.24 mmol) in

Ac₂O (5 ml) was added Zn powder (1.25 g). The reaction

mixture was stirred at room temperature and monitored by

TLC until complete. It was then filtered and concentrated in

vacuo. The crude residue was purified by flash chromatog-

NMR and ¹³C-NMR of Ib showed the presence of two

rotamers in CDCl₃and one in CD₃OD. ¹H NMR

(300 MHz, CDCl₃): δ 7.74 (d, J=7.4 Hz, Ar), 7.60 (m,

Ar), 7.50 (app t, J=7.8 Hz, Ar), 7.41–7.28 (m, Ar), 7.17 (d,

J=9.0 Hz, NH), 7.03 (d, J=9.9 Hz, NH), 6.29 (d, J=

9.6 Hz, NH), 6.00 (d, J=9.6 Hz, NH), 5.30–5.03 (m), 5.01

Glycoconj J (2011) 28:537–555

551

raphy (gradient of 0–80% EtOAc-Hexane over 60 min) to

afford 9a (1.14 g, 67%). ¹H NMR (300 MHz, CDCl₃)

δ7.77 (d, J=7.5 Hz, 2H, Ar), 7.66 (d=7.3 Hz, 2H, Ar),

7.43–7.29 (m, 9H, Ar), 5.82 (d, J=9.1 Hz, 1H, NH), 5.36

(d, J=8.4 Hz, 1H, NH), 5.24–5.18 (m, 4H, H-4, H-3,

OCH₂Ph), 4.64 (d, J=8.3 Hz, 1H, H-1), 4.48–4.23 (m, 5H,

α-H, β-H, FmocCH₂, FmocCH), 4.13–3.99 (m, 2H, H-6ª^,b),

3.89–3.79 (m, 1H, H-2), 3.70 (t, J=6.7 Hz, 1H, H-5), 2.13,

2.03, 2.02, 1.95 (4 s, each 3H, 4Ac), 1.22 (d, J=6.3 Hz, 3H,

Thr-CH₃), ESI HRMS: calcd for C₄₀H₄₄N₂O₁₃Na [M+Na]⁺

m/z, 783.2741; found, 783.5063.

calcd for C₄₀H₄₄N₂O₁₃Na [M+Na]⁺m/z, 783.2741; found,

783.5068.

N^α-fluoren-9-ylmethoxycarbonyl-O-(2-acetamido-2-deoxy-

3,4,6-tri-O-acetyl-β-D-glucopyranosyl)-L-threonine (Id)

To a solution of 9b (804 mg, 1.06 mmol) in methanol

(50 ml) was added 10% Pd/C (200 mg). The mixture was

stirred under one atmosphere of hydrogen gas for 1 h. The

catalyst was removed by filtration and after solvent

evaporation, the residue was purified by flash chromatog-

raphy using DCM as solvent A and 20% MeOH-DCM as

solvent B (gradient of 0–35% B over 25 min) to afford the

N^α-fluoren-9-ylmethoxycarbonyl-O-(2-acetamido-2-deoxy-

3,4,6-tri-O-acetyl-β-D-galactopyranosyl)-L-threonine (Ic)

To a solution of 9a (1 g, 1.32 mmol) in methanol (60 ml)

was added 10% Pd/C (250 mg). The mixture was stirred

under one atmosphere of hydrogen gas for 1 h. The catalyst

was removed by filtration and after solvent evaporation, the

residue was purified by flash chromatography using DCM

as solvent A and 20% MeOH-DCM as solvent B (gradient

titled compound Id (480 mg, 71%). H NMR (300 MHz,

1

CDCl₃): δ 7.76 (d, J=7.4 Hz, 2H, Ar), 7.64 (m, 2H, Ar),

7.42–7.28 (m, 4H, Ar), 6.06 (d, J=8.5 Hz, 1H, NH), 5.90

(d, J=9.0 Hz, 1H, NH), 5.29 (app t, J=8.0 Hz, 1H, H-3),

5.05 (t, J=9.6 Hz, 1H, H-4), 4.78 (d, J=8.3 Hz, 1H, H-1),

4.47–4.09 (m, 7H, β-H, FmocCH₂, H-6ª, FmocCH, α-H,

H-6^b), 3.84–3.75 (m, 1H, H-2), 3.72–3.64 (m, 1H, H-5),

2.07 (s, 3H, Ac), 2.01(s, 6H, 2Ac), 1.93 (s, 3H, Ac), 1.23

(d, J=7.0 Hz, 3H, Thr-CH₃).¹³C NMR (75 MHz, CDCl₃):

δ 172.6, 171.7, 170.9, 169.5 (COCH₃), 156.9 (CO Fmoc),

143.9, 143.7, 141.3, 127.7, 127.1, 125.2, 120.0 (Ar C), 99.4

(C-1), 75.6 (β-C), 72.1, 71.6 (C-3, C-5), 68.7 (C-4), 67.3,

61.9, 58.2, 54.8 (CH₂Fmoc, C-6, α-C, C-2), 47.1 (CH

Fmoc), 23.2, 20.8, 20.7, 20.6 (COCH₃), 17.6 (CH₃Thr).

The NMR data is in accord with the literature [48]. ESI

HRMS: calcd for C₃₃H₃₉N₂O₁₃[M+H]⁺m/z, 671.2452;

found, 671.4766.

1

of 0–80% B over 35 min) to afford Ic (680 mg, 77%). H

NMR (300 MHz, CD₃OD): δ 7.79 (d, J=7.3 Hz, 2H, Ar),

7.70 (m, 2H, Ar), 7.41–7.30 (m, 4H, Ar), 5.33 (d, J=

3.0 Hz, 1H, H-4), 5.08 (dd, J=3.4 Hz, J=11.3 Hz, 1H, H-

3), 4.60 (d, J=8.4 Hz, 1H, H-1), 4.44–4.34 (m, 3H, β-H,

FmocCH₂), 4.32–3.96 (m, 6H, FmocCH, α-H, H-6ª^,b, H-2,

H-5), 2.10, 2.01, 1.96, 1.95 (4 s, each 3H, 4Ac), 1.20 (d, J=

6.3 Hz, 3H, Thr-CH₃).¹³C NMR (75 MHz, CD₃OD): δ

172.5, 170.8, 170.7, 170.3 (COCH₃), 157.6 (CO Fmoc),

143.9, 143.7, 141.2, 127.4, 126.8, 124.9, 119.5 (Ar C),

100.2 (C-1), 75.4 (β-C), 70.5, 70.2 (C-3, C-5), 66.8 (C-4),

66.5, 60.9, 58.6 (CH₂Fmoc, C-6, α-C), 50.3 (C-2), 47.0

(CH Fmoc), 21.6, 19.2 (COCH₃), 16.6 (CH₃Thr). ESI

HRMS: calcd for C₃₃H₃₈N₂O₁₃Na [M+Na]⁺m/z,

693.2271; found, 693.4538.

N^α-fluoren-9-ylmethoxycarbonyl-O-(2,3,4,6-tetra-O-acetyl-

β-D-galactopyranosyl)-L-threonine benzyl ester (14a) A

mixture of Fmoc-Thr-OBn (1.82 g, 4.22 mmol), 13a

(3.25 g, 6.6 mmol) and activated 4A° molecular sieves in

dry CH₂Cl₂was stirred at room temperature for 30 min

under argon. After the addition of TMSOTf (15.38 μl,

0.0844 mmol) at 0°C, the reaction mixture was stirred at

room temperature until completion indicated by TLC. The

mixture was diluted with CH₂Cl₂and quenched with

triethylamine. The reaction mixture was filtered, washed

with saturated NaHCO₃, H₂O and dried over MgSO₄.

Purification by flash chromatography (gradient of 0–50%

EtOAc-Hexane over 40 min) afforded 14a (2.3 g, 72%) as a

white solid. ¹H NMR (300 MHz, CDCl₃) δ7.76 (d, J=

7.4 Hz, 2H, Ar), 7.63 (m, 2H, Ar), 7.41–7.28 (m, 9H, Ar),

5.68 (d, J=9.1 Hz, 1H, NH), 5.33 (d, J=2.9 Hz, 1H, H-4),

5.27–5.11 (m, 3H, OCH₂Ph, H-2), 4.95 (dd, J=3.4 Hz, J=

10.5 Hz, 1H, H-3), 4.48–4.33 (m, 5H, α-H, β-H, H-1,

FmocCH₂), 4.27–4.22 (m, 1H, FmocCH), 4.14–4.01 (m,

2H, H-6ª^,b), 3.72–3.67 (m, 1H, H-5), 2.13, 2.04, 2.03, 1.99

(4 s, each 3H, 4Ac), 1.22 (d, J=6.1 Hz, 3H, Thr-CH₃). ESI

HRMS: calcd for C₄₀H₄₃NO₁₄Na [M+Na]⁺m/z, 784.2580;

found, 784.5383.

N^α-fluoren-9-ylmethoxycarbonyl-O-(2-acetamido-2-deoxy-

3,4,6-tri-O-acetyl-β-D-glucopyranosyl)-L-threonine benzyl

ester (9b) A mixture of Fmoc-Thr-OBn (1.82 g, 4.22 mmol)

and 7b (3.95 g, mmol) was treated as described for the

preparation of 8a. The crude product 8b (3.5 g) was taken

for the next reaction. Compound 9b was prepared from 8b

as described for preparation of 9a. Purification of crude

residue by flash chromatography (gradient of 0–80%

EtOAc-Hexane over 40 min) afforded 9b (2.25 g, 70%)

as an amorphous white solid. ¹H NMR (300 MHz,

CD₃OD): δ 7.79 (d, J=7.5 Hz, 2H, Ar), 7.66 (d, J=

7.2 Hz, 2H, Ar), 7.40–7.25(m, 9H, Ar), 5.28–4.93 (m, 4H,

H-3, H-4, OCH₂Ph), 4.62 (d, J=8.4 Hz, 1H, H-1), 4.45–

4.19 (m, 6H, β-H, FmocCH₂, H-6ª, α-H, FmocCH), 3.96

(dd, J=2.0 Hz, J=12.2 Hz, H-6^b), 3.80 (dd, J=8.5 Hz, J=

10.4 Hz, 1H, H-2), 3.58 (m, 1H, H-5), 1.99, 1.93 (2 s, each

6H, 4Ac), 1.18 (d, J=6.3 Hz, 3H, Thr-CH₃), ESI HRMS:

552

Glycoconj J (2011) 28:537–555

N^α-fluoren-9-ylmethoxycarbonyl-O-(2,3,4,6-tetra-O-acetyl-

β-D-galactopyranosyl)-L-threonine (Ie) To a solution of

14a (1 g, 1.31 mmol) in methanol (50 ml) was added 10%

Pd/C (200 mg). The mixture was stirred under one

atmosphere of hydrogen gas for 1 h. The catalyst was

removed by filtration and after solvent evaporation, the

residue was purified by flash chromatography using DCM

as solvent A and 10% MeOH-DCM as solvent B (gradient

of 0–40% B over 35 min) afforded Ie (670 mg, 76%) as a

white crystalline solid. ¹H NMR (300 MHz, CD₃OD) δ7.78

(d, J=7.3 Hz, 2H, Ar), 7.68 (m, 2H, Ar), 7.41–7.29 (m, 4H,

Ar), 5.38 (d, J=2.4 Hz, 1H, H-4), 5.16–5.04 (m, 2H, H-2,

H-3), 4.64 (d, J=7.3 Hz, 1H, H-1), 4.42–3.99 (m, 8H, α-H,

β-H, FmocCH₂, FmocCH, H-6ª^,b, H-5), 2.09, 2.03, 2.00,

1.96 (4 s, each 3H, 4Ac), 1.20 (d, J=6.2 Hz, 3H, Thr-CH₃).

¹³C NMR (75 MHz, CD₃OD) δ 171.9, 170.7, 170.6, 170.2

(COCH₃), 170.1, 157.4 (CO Fmoc), 143.9, 143.7, 141.2,

127.4, 126.8, 125.0, 119.6 (Ar C), 99.4 (C-1), 75.43 (β-C),

70.83, 70.3, 69.1, 67.2 (C-3, C-5, C-2, C-4), 66.8, 60.7,

58.4 (CH₂Fmoc, C-6, α-C), 47.0 (CH Fmoc) 19.5, 19.2,

19.1 (COCH₃), 16.6 (CH₃Thr). ESI HRMS: calcd for

C₃₃H₃₈NO₁₄[M+H]⁺m/z, 672.2292; found, 672.4792.

α-H, β-H, FmocCH₂, FmocCH, H-6^a), 4.15 (dd, J=

4.1 Hz, J=12.2 Hz, 1H, H-6^b), 3.69–3.63 (m, 1H, H-5),

2.10, 2.05, 2.03, 2.02 (4 s, each 3H, 4Ac), 1.24 (d, J=

6.3 Hz, 3H, Thr-CH₃). ¹³C NMR (75 MHz, CDCl₃) δ

172.2, 171.8, 170.4, 169.5 (COCH₃), 169.3, 156.9 (CO

Fmoc), 143.9, 143.7, 141.3, 127.7, 127.1, 125.2, 119.9

(Ar C), 99.6 (C-1), 76.0 (β-C), 72.6, 71.7, 71.2, 68.4 (C-3,

C-5, C-2, C-4), 67.4, 61.7, 58.1 (CH₂Fmoc, C-6, α-C),

47.1 (CH Fmoc), 20.8, 20.6 (COCH₃), 17.6 (CH₃Thr).

ESI HRMS: calcd for C₃₃H₃₇NO₁₄[M+H]⁺m/z,

672.2292; found, 672.4753.

N^α-fluoren-9-ylmethoxycarbonyl-O-(2,3,4,6-tetra-O-acetyl-

α-D-mannopyranosyl)-L-threonine benzyl ester (17) To a

solution of completely dried Fmoc-Thr-OBn (2 g,

4.64 mmol) in dry CH₂Cl₂(20 mL) were added AgOTf

(1.67 g, 6.489 mmol) and flame dried 4 A° molecular

sieves. A solution of bromide 16 (3.247 g, 7.90 mmol) in

CH₂Cl₂(5 mL) was slowly added at 0°C under the constant

supply of argon gas. The reaction was stirred overnight at

room temp. and quenched with DIPEA (807 μL,

6.952 mmol). The reaction mixture was filtered through

celite. The filtrate was washed with conc. Na₂S₂O₃, conc.

Na₂CO₃and brine. All the aqueous washes were back

extracted with CH₂Cl₂, dried over MgSO₄, filtered, and

concentrated. The purification of the residue by flash

chromatography (gradient of 0–60% EtOAc-Hexane over

N^α-fluoren-9-ylmethoxycarbonyl-O-(2,3,4,6-tetra-O-acetyl-

β-D-glucopyranosyl)-L-threonine benzyl ester (14b) A

mixture of Fmoc-Thr-OBn (3.64 g, 8.44 mmol) and 13b

(5.41 g, 10.97 mmol) was treated as described for

synthesizing 14a. Purification of the crude mixture by flash

chromatography (gradient of 0–40% EtOAc-Hexane over

30 min) afforded 14b (5 g, 79%) as a white solid. ¹H NMR

(300 MHz, CDCl₃) δ7.76 (d, J=7.4 Hz, 2H, Ar), 7.57 (d, J=

7.1 Hz, 2H, Ar), 7.42–7.28 (m, 9H, Ar), 5.63 (d, J=9.2 Hz,

1H, NH), 5.29–5.11 (m, 3H, OCH₂Ph, H-2), 5.06–4.99

(t, J=9.6 Hz, 1H, H-3), 4.94–4.88 (m, 1H, H-4), 4.46–4.33

(m, 4H, α-H, β-H, FmocCH₂), 4.44 (d, J=8.2 Hz, 1H, H-1),

4.26–4.01 (m, 3H, FmocCH, H-6ª^,b) 3.49–3.43 (m, 1H, H-5),

2.04, 2.02, 2.01, 2.00 (4 s, each 3H, 4Ac), 1.22 (d, J=

6.3 Hz, 3H, Thr-CH₃). ESI HRMS: calcd for C₄₀H₄₃NO₁₄Na

[M+Na]⁺m/z, 784.2580; found, 784.4357.

1

40 min) afforded 17 (2.82 g, 80%). H NMR (300 MHz,

CDCl₃) δ 7.78 (d, J=7.4 Hz, 2H, Ar), 7.65 (d, J=7.3 Hz,

2H, Ar), 7.44–7.31 (m, 9H, Ar), 5.58 (d, J=9.6 Hz, 1H,

NH), 5.37–5.19 (m, 4H, H-3, H-4, OCH₂Ph), 5.07 (dd, J=

1.7 Hz, J=3.8 Hz, 1H, H-2), 4.86 (br s, 1H, H-1), 4.54–4.34

(m, 4H, α-H, β-H, FmocCH₂), 4.31–4.02 (m, 4H, FmocCH,

H-6ª^,b, H-5), 2.16, 2.08, 2.07, 2.02 (4 s, each 3H, 4Ac), 1.32

(d, J=6.4 Hz, 3H, Thr-CH₃). ESI HRMS calculated for

C₄₀H₄₃NO₁₄Na [M+Na]⁺m/z, 784.258; found, 784.442.

N^α-fluoren-9-ylmethoxycarbonyl-O-(2,3,4,6-tetra-O-α-D-

mannopyranosyl)-L-threonine (Ig) Compound 17 (1 g,

1.31 mmol) and 5%Pd/C (200 mg) were stirred in methanol

(50 mL) under H₂(1 atm) for 45 min. The catalyst was

filtered through celite and the filtrate was concentrated. The

residue was purified by flash chromatography using DCM

as solvent A and 10% MeOH-DCM as solvent B (gradient

of 0–60% B over 40 min) to yield pure Ig (625 mg, 71%).

¹H NMR (300 MHz, MeOD) δ7.80 (d, J=7.4 Hz, 2H, Ar),

7.73–7.70 (m, 2H, Ar), 7.42–7.29 (m, 4H, Ar), 5.30 (dd, J=

3.3 Hz, J=10.0 Hz, 1H, H-3), 5.24–5.17 (m, 2H, H-2, H-4),

4.97 (br s, 1H, H-1), 4.48–4.34 (m, 2H, α-H, β-H), 4.32–

4.08 (m, 6H, H-6ª, H-6^b, FmocCH₂, FmocCH, H-5), 2.11,

2.05, 2.04, 1.96 (4 s, each 3H, 4Ac), 1.30 (d, J=6.4, 3H,

Thr-CH₃). ¹³C NMR (75 MHz, CD₃OD) δ 170.9, 170.3,

170.2, 170.1 (COCH₃), 143.9, 143.9, 141.2, 127.4, 126.8,

N^α-fluoren-9-ylmethoxycarbonyl-O-(2,3,4,6-tetra-O-acetyl-

β-D-glucopyranosyl)-L-threonine (If) To a solution of 14b

(2.18 g, 2.86 mmol) in EtOAc (70 ml) was added 10%

Pd/C (1 g). The mixture was stirred under one atmosphere

of hydrogen gas until the reaction was complete. The

catalyst was removed by filtration and after solvent evapora-

tion, the residue was purified by flash chromatography using

DCM as solvent A and 20% MeOH-DCM as solvent B

(gradient of 0–60% B over 30 min) to afford compound If

1

(1.7 g, 88%). H NMR (300 MHz, CDCl₃): δ 7.77 (d, J=

7.4 Hz, 2H, Ar), 7.65 (m, 2H, Ar), 7.43–7.30 (m, 4H, Ar),

5.74 (d, J=9.4 Hz, 1H, NH), 5.24–4.94 (m, 3H, H-2, H-3,

H-4), 4.55 (d, J=7.9 Hz, 1H, H-1), 4.45–4.24 (m, 6H,

Glycoconj J (2011) 28:537–555

553

124.9, 124.9, 119.5 (Ar C), 98.9 (C-1), 77.1 (β-C), 69.4,

69.3, 68.9 (C-3, C-4, C-2), 66.8, 66.1, 62.4, 59.1 (C-5, α-C,

CH₂Fmoc, C-6), 47.0 (CH Fmoc), 19.2 (COCH₃), 17.5

(CH₃Thr). ESI HRMS: calcd for C₃₃H₃₇NO₁₄Na [M+Na]⁺

m/z, 694.2111; found, 694.4171.

The pellet was resuspended in 10 ml PBS, diluting the cells

to 2% of their packed volume. For each test condition,

90 μl of the erythrocytes were plated in a 96-well microtiter

plate (Greiner Bio-one, Wemmel, Belgium) along with

10 μl of peptide solution serially diluted in the same buffer

with a starting concentration of 100 μM added to the wells

and the plate was incubated at 37°C for 90 min. Any

hemolytic activity would manifest as rupture of RBCs and

release of hemoglobin into the solution. The resuspension

buffer, PBS, was used as negative control while RBCs

resuspended in 1% TritonX-100, which results in their

complete lysis, was used as positive control. Subsequently,

the plate was centrifuged at 4,000 rpm for 10 min to pellet intact

cells and 75 μl of supernatant was transferred to a flat bottom

polystyrene microtiter plate (Greiner Bio-one, Wemmel,

Belgium). Its absorbance was read at 405 nm and the

percentage of RBC lysis was calculated. The hemolysis

affected by only PBS and 1% TritonX-100 was considered as

negative control and 100% hemolysis, respectively. Each test

condition was set up in duplicate and the results have been

averaged from three independent experiments.

Peptide synthesis All peptides used were synthesized by

the solid-phase method using an automated peptide synthe-

sizer (433A; Applied Biosystems Inc.), employing Fmoc-

methodology. The peptides were cleaved from the resin by

treatment with TFA/thioanisole/phenol/water/EDT in ratio

as recommended by Applied Biosystems Inc. The crude

peptides were purified using C-18 column (Deltapak -100

Å, 15μ, spherical, 19x300mm, Waters). The sugar residue

of purified glycosylated peptide was deacetylated by

treatment with 5% hydrazine-hydrate and finally again

purified by reverse phase column chromatography using C-18

column. The synthesized peptides were determined to

be >95% pure by HPLC. Molecular mass determinations were

made by using MALDI TOF/TOF 4800 (Applied Biosystems

MDS SCIEX) with α- cyano-4-hydroxycinnamic acid as the

matrix and ESI-MS system (Applied Biosystems Mariner

System 5220) both operating in positive mode.

MTT assay The cytotoxic potential of synthesized peptides

was determined following incubation of rat myeloma SP2/O

(1×10⁵cell/ml) cell line using the MTT assay. This method is

based on the reduction of the salt, methylthiazolyldiphenyl-

tetrazolium bromide (MTT) into a crystalline blue formazan

product by the cellular oxidoreductases of viable cells.

Following 24 h incubation, cells were washed and then

incubated with fresh culture media and 50 μl MTT (5 mg/ml)

in 0.1 M PBS, pH 7.4 at 37°C in a humid atmosphere with

5% CO₂for 4 h. Media were then gently aspirated from test

cultures and 50 μl of dimethyl sulfoxide (DMSO) was added

to all wells. The plates were then shaken for 2 min and the

absorbance was read at 540 nm in a microtiter plate reader.

The percentage of viability was calculated as AT/AC×100;

where AT and AC are the absorbances of treated and control

cells, respectively. In the positive and negative controls the

peptide solution was substituted by the same volumes of

ethanol and PBS, respectively. Each test condition was set up

in triplicate and the results have been averaged from three

independent experiments.

Antibacterial activity assay by radial diffusion The anti-

bacterial activity of all the peptides was ascertained by a

radial diffusion assay using double-layered agarose as

described previously [49]. The bacterial strains used for

this experiment were: Escherichia coli ATCC25922,

Escherichia coli BL21λD3, Salmonella typhi Vi⁺, and

Salmonella typhimurium. The 5 mM peptide solution

corresponding to amount of 25 nmoles of each peptide

was used to determine the comparative antibacterial

activity. The assay was done in triplicate and repeated

thrice to calculate the average and the standard error.

Circular-Dichroism spectroscopy The circular-dichroism

(CD) spectra of all peptides were obtained at 25°C in a

1 mm pathlength cuvette in the range of 250–190 nm using

CD spectrometer (J-815 JASCO Corporation, Japan) with a

1.0 nm bandwith at 0.1 nm resolution and 1.0 s of response

time. 5 scans with a speed of 200 nm/min were accumu-

lated and averaged. The spectra were recorded in distilled

water, 10 mM SDS in 10 mM PB, pH 7.2, 90% TFE in

water. Results were expressed as mean residue ellipticity in

[θ] (Deg-cm²/dmol).

Acknowledgements This work was supported by grant from the

Department of Biotechnology, Government of India.

Hemolytic activity assay The hemolytic response was

measured spectrophotometrically by analyzing % lysis of

freshly isolated murine red blood cells [50]. A 200 μl

aliquot of packed erythrocyte volume was washed thrice

with cold phosphate-saline buffer (10 mM PB, pH 7.4,

150 mM NaCl) by centrifugation for 5 min at 1,150 g, 4°C.

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Article Doi

Glycosylated analogs of formaecin I and drosocin exhibit differential pattern of antibacterial activity

DOI: 10.1007/s10719-011-9353-2

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